Enzyme-based electrochemical sensors are widely used in the detection and measurement of analytes in clinical, environmental, agricultural and biotechnological applications. Analytes that can be measured in clinical assays of fluids of the human body include, for example, glucose, lactate, cholesterol, bilirubin and amino acids. Levels of these analytes in biological fluids, such as blood, are important for the diagnosis and the monitoring of diseases, such as diabetes mellitus, a disorder of the pancreas where insufficient production of insulin prevents normal regulation of blood sugar levels.
Small volume (e.g., less than 0.5 microliter), in vitro, electrochemical sensors are used with FREESTYLE® and FREESTYLE FLASH® glucose meters strips manufactured by Abbott Diabetes Care Inc., Alameda, Calif., USA. These test strip sensors generally include a working electrode on a first substrate, a counter (or counter/reference) electrode on a second substrate, and a sample chamber. The sample chamber is configured so that when a sample (e.g., of blood) is provided in the chamber, the sample is in electrolytic contact with both the working electrode, the counter electrode and any reference electrodes or indicator electrodes that may be present. This allows electrical current to flow between the electrodes to effect the electrolysis (electrooxidation or electroreduction) of the analyte. A spacer is generally positioned between first substrate and second substrate to provide a spacing between electrodes and to provide the sample chamber in which the sample to be evaluated is housed.
The electrode system (carbon, gold, platinum, etc.) is laid on an insulating substrate surmounted by a reagent layer containing at least a redox enzyme/cofactor (GDH/NAD, HBDH/NAD, GOx/FAD, GDH/PQQ, GDH/FAD) which acts on an analyte (glucose, 3-hydroxybutyrate, etc.) and a redox mediator which provides electrical communication between the enzyme/cofactor and the electrode. The action of enzyme/cofactor on the analyte results in the conversion of oxidized mediator to its reduced form which, in turn, is oxidized at an electrode. This generates an electrical signal which is proportional to the analyte concentration. The enzyme is specific to the analyte to be detected, or to a product of the analyte. The turnover rate of the enzyme is typically related (preferably, but not necessarily, linearly) to the concentration of the analyte itself, or to its product, in the test solution.
The first generation of electrochemical biosensors used oxygen as the electron acceptor. Oxygen, however, becomes a limiting factor in the enzymatic reaction at high substrate concentrations due to its limited solubility in liquid. This limits the upper linear range of the oxidase-based biosensors. In order to overcome problems associated with insufficient oxygen concentration, a second generation of biosensors was developed that used electron or “redox” mediators as substitutes for oxygen. Such electron mediators, also referred to as “transition metal complexes” are redox chemicals capable of mediating the electron transfer in the regeneration of the enzyme to the active form. In some cases the transition metal complexes are coordination complexes comprising osmium, for example. The linear range of the resulting biosensors was significantly improved.
A redox mediator assists in the electrical communication between the working electrode and the enzyme. The redox mediator can be dissolved in the fluid to be analyzed, which is in electrolytic contact with the electrodes, or can be applied within a coating on the working electrode in electrolytic contact with the analyzed solution. The coating is preferably not soluble in water, though it may swell in water. Useful devices can be made, for example, by coating an electrode with a film that includes a redox mediator and an enzyme where the enzyme is catalytically specific to the desired analyte, or its product. In contrast to a coated redox mediator, a diffusional redox mediator, which can be soluble or insoluble in water, functions by shuttling electrons between, for example, the enzyme and the electrode. In any case, when the substrate of the enzyme is electrooxidized, the redox mediator transports electrons from the substrate-reduced enzyme to the electrode; and when the substrate is electroreduced, the redox mediator transports electrons from the electrode to the substrate-oxidized enzyme.
Recent enzyme-based electrochemical sensors have employed a number of different redox mediators such as monomeric ferrocenes, quinoid compounds including quinines (e.g., benzoquinones), nickel cyclamates, and ruthenium amines. For the most part, these redox mediators have one or more of the following limitations: the solubility of the redox mediators in the test solutions is low, their chemical, light, thermal, and/or pH stability is poor, or they do not exchange electrons rapidly enough with the enzyme or the electrode or both. Some mediators with advantageous properties are difficult to synthesize. Additionally, the redox potentials of some of these reported redox mediators are so oxidizing that at the potential at which the reduced mediator is electrooxidized on the electrode, solution components other than the analyte are also electrooxidized. Some other of these reported redox mediators are so reducing that solution components, such as, for example, dissolved oxygen, are also rapidly electroreduced. As a result, the sensor utilizing the mediator is not sufficiently specific.
Accordingly, although the mediator/oxidase-based biosensors eliminate the dependence on the oxygen concentration for the extended linear range of the sensor, oxygen-related drawbacks still exist. For example, some mediators are not as efficient at shuttling electrons with the enzyme as the oxygen molecule. And, any oxygen in the sample solution can effectively compete for the enzyme site. Thus, mediator/oxidase-based biosensors may generate inaccuracies resulting from different oxygen concentrations. This becomes more serious when the substrate concentration is at a low concentration level (e.g., glucose concentration less than 70 mg/dL).
To obviate the interference resulting from the varying oxygen concentration or so-called “oxygen effect” described above using the mediator/oxidase-based biosensors, an oxygen-insensitive enzyme such as glucose dehydrogenase (GDH) was used to replace the oxygen-sensitive oxidase such as glucose oxidase. Flavin adenine dinucleotide (FAD) and pyrroloquinoline quinone (PQQ) are redox coenzymes (also referred to as “co-factors”) that act as prosthetic groups, which are involved in catalyzing reactions that generate electrons from the oxidation of glucose and other sugars. Glucose dehydrogenase, whose coenzyme can be PQQ or FAD, does not interact with oxygen. Therefore, the resultant glucose sensor is unaffected by variable oxygen concentration in the sample. Such a sensor may be in the form of a strip-commonly referred to as a test strip or sensor strip, e.g., analogous to FREESTYLE® strips manufactured by Abbott Diabetes Care Inc., Alameda, Calif., USA. FREESTYLE® is a product that has been developed and marketed using glucose dehydrogenase.
The use of glucose dehydrogenase overcomes the problems associated with the oxygen effect. Glucose dehydrogenase, however, is not as specific as glucose oxidase. Glucose dehydrogenase reacts not only with glucose but with sugars that are structurally similar to glucose such as galactose and maltose. Maltose is composed of two glucose units and galactose differs in structure from glucose only in the position of the hydroxyl group on carbon number 4; accordingly, significant interference may be expected. Indeed, glucose dehydrogenase-based biosensors are sensitive to maltose and have difficulty discriminating between glucose and galactose. Cross-reactivity with maltose is particularly important due to the use of icodextrin in peritoneal dialysis, which metabolizes to maltose in vivo. If a glucose monitor or test strip uses a glucose dehydrogenase pyrroloquinolinequinone method (PQQ-GDH), a falsely high glucose reading may be obtained, potentially causing over-administration of insulin to a subject, thereby unnecessarily lowering blood glucose levels. Such a lowering of blood glucose levels may cause serious reactions in patients such as, but not limited to, loss of consciousness.
Responding to the need for a glucose biosensor that does not “cross-react” with unintended analytes, and which can be used safely with peritoneal dialysis patients, FAD-GDH has replaced PQQ-GDH in some systems described herein. Unlike PQQ-GDH, FAD-GDH does not catalyze reactions that generate electrons from maltose or galactose. It is therefore a desirable choice as the coenzyme for use with glucose dehydrogenase.
The FAD-GDH enzyme system, however, performs ineffectively with certain mediators. The mediator known as “nPBI” having an n-pentylbenzimidazole ligand, shown below, for example, does not compete favorably for electrons from the enzyme when FAD is used in concert with glucose dehydrogenase.
